Evaluation of the amino acid binding site of Mycobacterium tuberculosis glutamine synthetase for drug discovery

Article abstract of DOI:10.1016/j.bmc.2008.04.015

A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature

Full text of DOI:10.1016/j.bmc.2008.04.015

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5501–5513
Evaluation of the amino acid binding site of Mycobacterium
tuberculosis glutamine synthetase for drug discovery
Anneli Nordqvist,a Mikael T. Nilsson,b Svenja Ro¨ttger,a, Luke R. Odell,a
Wojciech W. Krajewski,b C. Evalena Andersson,b Mats Larhed,a
a,
Sherry L. Mowbrayc, and Anders Karlen
*
*
´
aDivision of Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Uppsala University, Biomedical Centre,
Box 574, SE-751 23 Uppsala, Sweden
bDepartment of Cell and Molecular Biology, Uppsala University, Biomedical Centre, Box 596, SE-751 24 Uppsala, Sweden
cDepartment of Molecular Biology, Swedish University of Agricultural Sciences,
Biomedical Centre, Box 590, SE-751 24 Uppsala, Sweden
Received 18 October 2007; revised 1 April 2008; accepted 8 April 2008
Available online 11 April 2008
Abstract—A combination of a literature survey, structure-based virtual screening and synthesis of a small library was performed to
identify hits to the potential antimycobacterial drug target, glutamine synthetase. The best inhibitor identified from the literature
survey was (2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4, IC50 of 610 15 lM). In the virtual screening 46,400 compounds were
docked and subjected to a pharmacophore search. Of these compounds, 29 were purchased and tested in a biological assay, allowing
three novel inhibitors containing an aromatic scaffold to be identified. Based on one of the hits from the virtual screening a small
library of 15 analogues was synthesized producing four compounds that inhibited glutamine synthetase.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
with concurrent hydrolysis of adenosine triphosphate
(ATP). The reaction passes through a phosphorylated
tetrahedral intermediate.3 GS is important in bacterial
nitrogen metabolism and the synthesized L-glutamine
is also a major component of the cell wall of
pathogenic mycobacteria.4 The potential of Mycobacte-
rium tuberculosis GS as a drug target was established in
a transposon mutagenesis study of strain H37Rv,
classifying MtGS as essential for optimal growth.5 Fur-
thermore, it has been shown that an antisense oligode-
oxyribonucleotide directed at the glnA1 gene, encoding
MtGS, reduced bacterial replication and the amount of
poly-L-glutamate/glutamine in the cell wall.6 Finally,
glnA1 has been found to be essential for M. tuberculo-
sis virulence.7 The two most active known GS inhibi-
tors described in the literature, L-methionine-(S)-
sulfoximine (MSO, 1, Chart 1) and phosphinothricin
(PPT, 2, Chart 1), have a profound effect on the
growth of M. tuberculosis.8 These are also the only
known inhibitors of MtGS. MSO inhibits growth of
M. tuberculosis in culture and within human mononu-
clear phagocytes8 and the L-(SR)-diastereomer has also
been demonstrated to have in vivo activity in a guinea
pig model.9
Tuberculosis (TB) is one of the most serious infectious
diseases. In 2005 the World Health Organization
(WHO) estimated that 1.6 million deaths were caused
by TB worldwide (www.who.int). Although the recom-
mended Directly Observed Treatment Short-course
therapy (DOTS) provides a cure for TB in many cases,
there is a great need for new more effective TB drugs
to combat multidrug-resistant tuberculosis and to short-
en the lengthy treatment time.1,2 Special effort is needed
to find and develop lead compounds acting against
already validated TB drug targets.2
Glutamine synthetase (GS, EC 6.3.1.2) catalyzes the
synthesis of glutamine from glutamate and ammonia
Keywords: Virtual screening; Glutamine synthetase; c-Glutamyl:ammonia
ligase.
*
Corresponding authors. Tel.: +46 18 471 4990; fax: +46 18 53 69 71
(S.L.M.); tel.: +46 18 471 4293; fax: +46 18 471 4474 (A.K.); e-mail
addresses: mowbray@xray.bmc.uu.se; anders.karlen@orgfarm.uu.se
˚
Present address: Caliber Synthesis AS, Skrautvalsvegen 77, N-2900
Fagernes, Norway.
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.015

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A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
binding to the amino acid site was optimized by using
relatively high concentrations of ATP and magnesium
ions, and choosing a glutamate concentration similar
to the Km for that substrate. DMSO, used for dissolving
some inhibitors, was found to have an activating effect
on MtGS. Approximately 10% activation was observed
at a DMSO concentration of 2% (v/v). However, the in-
crease in activity could be corrected for by including
DMSO in the compound-free reaction as well as in the
background reaction. That the correction for the effect
of DMSO did not interfere with the inhibition analysis
was demonstrated by tests with compound 4, which
was dissolved in both MilliQ water and DMSO, and
found to have a percentage of inhibition of 58 16%
and 56 10%, respectively.
Chart 1.
Amino acid analogues of MSO, PPT and mimics of the
phosphorylated intermediate have been synthesized and
evaluated on GS from different species in a number of
studies.10–29 Much effort has been invested in developing
GS inhibitors not only for antibacterial research, but
also for weed control,20,25,30–32 since GS is a key enzyme
in ammonia assimilation in plants. However, no inhibi-
tor has shown improved activity compared to MSO or
PPT. One of the most active PPT analogues (3, Chart
1) had a Ki in the same range as PPT when tested against
GS isolated from Escherichia coli, spinach leaves and
tobacco cultured cells.19,20 Other unnatural a-amino
acids have also been shown to exert inhibitory effects
on GS.31,33–36 Two such examples are (2S,5R)-2,6-diami-
no-5-hydroxyhexanoic acid (4, Chart 1, Ki 40 lM for
Salmonella typhimurium GS,29 and also inhibiting sheep
brain and soybean GS35) and L-2-amino-4-hydroxyam-
inobutyric acid (5, Chart 1, Ki 7 lM and 21 lM for sheep
brain and soybean GS,35 respectively). Additionally, a
set of aminomethylene-bisphosphonic acid derivatives
inhibit rice GS.32 It should be noted that prokaryotic
GSs are distantly related to eukaryotic ones, and that
inhibition patterns may not be directly comparable.
2.2. Literature survey
Since the residues in the amino acid binding site are
highly conserved,29 we anticipated that known inhibitors
of GS from other species, including distantly related GS
types, could also be active on MtGS. In fact a number of
inhibitors, including MSO,8 or its diastereomeric mixture
L-(SR),8,11,13,23 PPT,8,19,20,22–25,28 3,19,20 and 429,35 have
shown inhibitory activity when tested on both bacterial
and eukaryotic GSs. Therefore a literature search was
performed to identify known GS inhibitors for both
eukaryotic and prokaryotic enzymes. The majority of
the inhibitors reported previously were amino acid based
and had activities ranging from lM to mM. Compounds
that we could identify as commercially available (1–2, 4
and 6–8, Table 1) were purchased. Furthermore, the dia-
stereomer of 4 (compound 11) was synthesized. Another
compound (5) described in the literature, which inhibits
both sheep brain GS and soybean GS with similar po-
tency to that of MSO,35 was also synthesized, as well
as the analogue having a methyl group in the same posi-
tion as MSO and PPT (12). Finally, three analogues (9,
10 and 13) close to these were purchased.
Hit identification, that is the process of finding small-
molecule inhibitors of a target protein, is a key step in
drug discovery. Hit identification strategies37,38 include
literature surveys, identification of endogenous ligands
or active natural products, high-throughput screening,
virtual screening, chemogenomics, ligand design and
combinatorial chemistry. This study aims to find new
hits against the antimycobacterial drug target MtGS uti-
lizing three of the above mentioned hit identification
strategies. This work is focused on the amino acid site,
where all of the presently known inhibitors bind. First
the literature was surveyed to identify small molecules
known to be active on GS in other species that could
be purchased or easily synthesized. In parallel, a struc-
ture-based virtual screening of the amino acid binding
site was performed to uncover possible novel hits. Based
on the virtual screening results, a small library of ana-
logues was synthesized. Herein, we present the results
of our efforts.
The compounds above were screened for inhibitory
activity against MtGS at a single concentration (in gen-
eral 1.0 mM) using a biosynthetic assay. Inhibition of
MtGS was determined by comparison to that of unin-
hibited enzyme. The stereochemistry of the tested com-
pounds was mostly the pure L-form (1, 5–7, 9–10 and
12–13). PPT (2) was tested as a racemic mixture and 8
was tested as L-(SR)-sulfoxide. Compounds 4 and 11
were synthesized with defined stereochemistry at both
stereocenters as described in the experimental section.
Furthermore, the compounds were docked to the active
site of GS in an attempt to rationalize the structure–
activity relationship.
The most active inhibitor of S. typhimurium GS,29 com-
pound (4), was also found to inhibit MtGS. According
to the proposed binding mode from the docking calcula-
tion, 4 retains all the important interactions to Arg329,
His276, Gly272 and Glu135 in the amino acid backbone
that are found in the complex structure of MSO with
MtGS (Fig. 1). The 5-aminomethyl group approaches
the proposed ammonium ion binding site39 (residues
Glu219, Tyr186, Asp540 and Ser570, Fig. 1). Combined,
2. Results and discussion
2.1. Biological assay
A GS assay was developed based on the biosynthetic
reaction, considered to be the most biologically rele-
vant.29 The sensitivity of the present assay for inhibitors

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
Table 1. Compounds suggested by literature surveys
5503
COOH
H2N
R
Compound
R@
M. tuberculosisa
E. coli
S. typhimurium
Sheep brain
Soybean
O
S
NH
OH
1
81 3b
MSO
O
P
2
4
76 7b,c
PPT
58 16b
56 10d
Ki = 40 lM29
22 (0.5 mM)35
21 (0.5 mM)35
OH
NH2
OH
NH
5
6
7
26 14b
Ki = 7 lM35
Ki = 21 lM35
O
NIb
Kis = 54 lM17
P
OH
OH
O
33 13b
67 (25 mM)11
S
O
O
S
8
9
NIb,c
NIb
11–14 (25 mM)11
OH
10
NIb
OH
OH
NH2
OH
11
12
13
NIb
NIb
NId
N
O
S
O
OH
Compounds 1 (MSO) and 2 (PPT) are known MtGS inhibitors, while 4–8 are known inhibitors of GSs of other species. Compounds 9–13 are
analogues of previously known inhibitors. Results for MtGS are reported as percent inhibition in the biosynthetic assay in the presence of 1 mM
compound. Literature values for GSs from other species were based on the transferase assay and are reported either as percent inhibition (where the
concentration of compound is given in parentheses), or where available, as Ki. In the transferase assay, glutamine and hydroxylamine are converted
into c-glutamyl hydroxamate and ammonia, in the presence of ADP, manganese and arsenate.
a Reported values for MtGS are an average from three independent experiments, reported together with the standard deviation. NI indicates no
inhibition. Substrate concentrations in the assay were 1 mM ATP, 10 mM L-glutamate and 30 mM ammonium chloride.
b Dissolved in H2O.
c Tested as a racemic mixture or a mixture of two diastereomers.
d Dissolved in DMSO.
these interactions could explain the relatively good inhi-
bition. The stereochemistry of the hydroxyl group of 4 is
required to be R, since the S-isomer (11) was inactive.
We therefore suggest that the interaction of the hydroxyl
group with Arg368 is important, since the inactive com-
pound (11) lacks this hydrogen bond (Fig. 1). An alter-
native explanation to the inactivity of 11 could be that
this position must be occupied by an oxygen atom.
The importance of the stereochemistry at the same posi-
tion in the active site has also previously been reported
for the sulfoximine group, with the L-(S)-enantiomer
of MSO being a more potent inhibitor of sheep brain
GS40 and E. coli GS.41
Because of the strong inhibitory activity of 5 with regard
to GS from other species (vide supra) we resynthesized

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A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
Figure 2. IC50 determination. Experimental data are shown for MSO
(1, triangles), PPT (2, squares) and (2S,5R)-2,6-diamino-5-hydroxy-
hexanoic acid (4, circles). Data points were normalized to ease
comparison, using the experimentally determined Hi and Lo values,
such that normalized fractional activity = (Y ꢁ Lo)/ (Hi ꢁ Lo). The
accompanying curves are those calculated from Eq. 1 using the
determined IC50 values (1, 51 lM, n = 3; 2, 1.9 lM, n = 3; 4, 610 lM,
n = 5) together with Hi = 1 and Lo = 0. Substrate concentrations used
in the assay were 1 mM ATP, 10 mM L-glutamate and 30 mM
ammonium chloride.
Figure 1. Active site of MtGS. Compounds 4 (black carbon atoms)
and 11 (magenta) docked in the amino acid binding site of MtGS,
compared to the crystal structure of phosphorylated L-methionine-
(S)-sulfoximine (green). Hydrogen bonds between 4 and the enzyme
are indicated with dotted lines.
this compound and evaluated its inhibitory potency
against MtGS. Unfortunately, compound 5 was only
weakly potent. The analogue having a terminal methyl
group corresponding to the methyl group in the sulfoxim-
ine and phosphinyl moiety of MSO and PPT, respectively,
was also synthesized (12). However, this compound
lacked inhibitory activity. Exchanging the hydroxylamine
nitrogen of 5 with a carbon generates the inactive com-
pound 9. Thus an oxygen atom at the 5-position of an
amino acid is not the only characteristic of an active com-
pound, although this has been identified as a common
structural feature in many known inhibitors.35 L-Homo-
serine (10), with a carbon chain that is one methylene unit
shorter than 9, was also tested and found to be inactive.
nitrogen and/or double bonded oxygen(s)) seem to be very
important for inhibition of the MtGS enzyme.
The IC50 values were determined for the most potent
compound (4) and for the two previously known inhib-
itors (1 and 2). MSO and PPT were the best inhibitors,
with IC50 values of 51 6 lM and 1.9 0.4 lM, respec-
tively (Fig. 2). Inhibitor 4 had an IC50 value of 610 15
lM, which is 12 times higher than that of MSO. In total
the literature survey identified three new inhibitors of
MtGS among the tested compounds.
2.3. Virtual screening
Compound 6 has previously been reported to inhibit
E. coli GS (Kis 54 lM)17 but was found to be inactive
against MtGS. This finding was verified using a slightly
different biosynthetic assay (for experimental details, see
Lagerlund et al.42) where the activity of 6 was evaluated
on GS from both species. Compound 6 was fairly potent
on E. coli GS (61% inhibition at 1.1 mM), but lacked
inhibitory potency on MtGS. Thus, despite the con-
served residues in the amino acid binding site, there
are differences in the catalytic properties of E. coli GS
and MtGS that are reflected in the different inhibition
pattern seen for this compound.
Starting from a database of 2.1 million commercially
available compounds, the database was reduced to
46,400 compounds with a molecular weight <300 g/
mol containing carboxylic acid bioisosters43 (substrate),
amides (product) or functional groups that have previ-
ously been used as substitutes for the sulfoximine group
of MSO29 (Fig. 3). A docking protocol with FlexX was
used that successfully reproduced the binding of phos-
phorylated MSO (MSO-P) observed in the crystallo-
graphic structure of MtGS44 as the top scoring pose.
When non-phosphorylated MSO was docked, the top
scoring pose overlaid well with the equivalent portions
of MSO-P. PPT was also used to evaluate the docking
protocol. Its top scoring pose was generally in agree-
ment with the complex structure of S. typhimurium with
PPT,45 with the only difference being that the methyl
group is rotated by ꢀ180ꢁ. This is in accordance with
the binding mode of PPT suggested by Krajewski et
al. on the basis of high-resolution data.44 The 46,400
The sulfonic acid (13) did not show any inhibition. How-
ever, the corresponding sulfone (7) inhibited MtGS, with
33% inhibition at a concentration of 1.0 mM. The same
pattern was observed when comparing results with PPT
and 6. Additionally, the (SR)-sulfoxide (8) did not show
any inhibition. Thus, the structural features including a
methyl group together with two anchor points (imine

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
Table 2. Compounds derived from the virtual screening
5505
Compounda
% Inhibitionb
H
N
14
15
24 3c
O
N
H
O
S
NH2
O
O
26 13c
N
HOOC
N
H
N
NH
6
n = 1, NId
n = 2, NIc,f
n = 3, NId
n = 4, NId
COOH
n=1-4
O
16
17
18
H2N
n
P
OH
HO
OH
O
P
OH
19
NId,g
HOOC
NH2
NId,e
O
HOOC
H2N
20
P
OH
OH
HOOC
H2N
21
22
NId
NH2
Figure 3. Virtual screening flowchart. The 2D substructures were used
in primary filtering of the database. The pharmacophore with one
hydrogen bond donor (DON) and two hydrogen bond acceptors
(ACC) was used to select only the compounds where the first docking
solution was placed deep inside the amino acid binding site with a
hydrogen bonding pattern similar to L-methionine-(S)-sulfoximine (1).
O
NId
O
O
P
OH
H2N
OH
HOOC
HN
OH
O
compounds were then subjected to ligand pre-treatment
and docked in the amino acid binding site. The top scor-
ing poses were filtered using a rigid pharmacophore
search (Fig. 3), which yielded 3511 compounds that were
visually inspected. Among these, 29 compounds were se-
lected, purchased and screened for activity against
MtGS using the biosynthetic assay at a single concentra-
tion, in general 1.0 mM, The most interesting of these
are presented in Table 2.
23
48 16d
S
O
OH
Inhibition of MtGS is reported as percent at a compound concentra-
tion of 1.0 mM, as compared to uninhibited enzyme. Substrate con-
centrations in the assay were 1 mM ATP, 10 mM L-glutamate and
30 mM ammonium chloride. NI indicates no inhibition.
a Compounds 6, 16–18 were tested as L-amino acids and 19–22 as
racemic mixtures.
b Values are an average from three independent experiments, reported
together with the standard deviation.
The virtual screening approach identified three active
hits 14, 15 and 23 with 24%, 26% and 48% inhibition,
respectively (Table 2). Interestingly these are not related
to any previously known inhibitors of GS. The FlexX
docking results suggested that compound 14 fits in the
amino acid binding site (Fig. 4A) with the urea function-
ality facing Arg329 and Gly272. It places the tetrahedral
sulfonamide group at the position of the sulfoximine of
MSO-P, fulfilling one criterion possibly important for
an active compound (vide supra). The sulfonamide func-
tionality has also been used in an amino acid analogue
that inhibited E. coli GS.17 Both compounds 15 (Fig.
4B) and 23 (Fig. 4C) interact with the carboxyl group
in the same manner as MSO, forming an ionic interac-
tion and hydrogen bonds to Arg329 and His276.
Compound 23 lacks the backbone amino group, how-
c Dissolved in DMSO.
d Dissolved in H2O.
e Concentration in assay 0.20 mM.
f Concentration in assay 0.75 mM.
g Concentration in assay 0.93 mM.
ever, the secondary aromatic amino group instead forms
hydrogen bonds to Glu135. Compound 15 extends
somewhat further than MSO and can find additional
interactions with its heterocyclic ring higher up in the
amino acid binding site. All three compounds form a
hydrogen bond between the key residue Arg368 (vide
supra) and the double bonded oxygen of the sulfon-
amide, amide or sulfonic acid group, respectively.

5506
A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
Figure 4. Hits from the virtual screening. The three inhibitors identified in the virtual screening and the docked solution of these in the amino acid
binding site. Compounds 14 (A) and 15 (B) and 23 (C) show key interactions with residues in the binding site.
Glutamate analogues with a longer carbon chain have
been reported as substrates of sheep brain GS46,47 and
substituted glutamate analogues have been successfully
converted to active PPT analogues.23–27 However, none
of the 2-amino-phosphono-carboxylic acids (16– 18)
identified in the virtual screening showed any inhibitory
activity, despite the fact that the phosphono group of
docked 2-amino-6-phosphonohexanoic acid (17) over-
laps well with the phosphono group of MSO-P. This is
in accordance with the results for DL-homophosphino-
thricin, which did not show any inhibition of sheep brain
GS.23 Thus the patterns of inhibition observed here agree
well between the prokaryotic and eukaryotic GSs.
It was also realized that compound (21) carrying the
ortho-substituted aminomethyl group could be obtained
using this route. Starting from 2-cyanobenzylbromide,
benzyliodide 42 was prepared in four steps. Phosphoryla-
tion of 42 with triethylphosphite gave the diethyl pro-
tected phosphate 43, which was treated with 9 M HCl to
afford the desired compound 20. In addition, the corre-
sponding methyl ester 22 was also isolated. It is believed
to have been formed via a transesterification with MeOH
in the first step of the synthesis of 20.
Compound 20 was also inactive even though it can
adopt a conformation that overlaps well with MSO-P.
Considering the results with 20 and the 2-amino-phos-
phono-carboxylic acids (16– 18), the tetrahedral sulfox-
imine group or a similar moiety four bonds distant from
the carbonyl carbon seems to be important for inhibi-
tory activity. The ortho-substituted aminomethyl com-
pound (21), the methyl ester (22), obtained from the
synthesis of 20, and the purchased para-substituted ana-
logue (19) did not inhibit MtGS.
In addition to the 29 compounds that were selected, the
ortho-substituted phenylalanine derivative 20 was identi-
fied as a promising compound, for which the amino acid
backbone and the phosphono group overlaid well with
the corresponding functional groups of MSO-P. How-
ever, it could not be purchased and was therefore synthe-
sized based on the method of Dorville et al. (Scheme 1).48
2.4. Synthesis of a small library
Compound 23, identified in the virtual screening, also
served as the starting point for a small synthetic library
(Table 3). Several functional groups, including a carbox-
ylic acid, a thioether, a sulfoxide, a sulfonamide and a
phosphonate ester, were investigated as R2 substituents.
The compounds were synthesized from glycine (24– 28)
or from L-(29– 33) or D-serine (34– 38), as described in
the experimental section. The desired products were pre-
pared via a CuI catalyzed N-arylation of glycine, D- and
L-serine, using K2CO3 as a base, with an appropriately
substituted arylbromide (Scheme 2).49 After work-up
the products were purified by preparative HPLC. Chiral
HPLC analysis was used to determine the chiral purity
(>95%) of the products.
The compounds were screened for activity against MtGS
at a single concentration, in general 1.0 mM, using the
biosynthetic assay. Four weak inhibitors were identified,
with 28, a phosphonate ester being the most potent. The
majority of the compounds displayed an apparent activa-
tion of the enzyme. Additional studies will be required to
establish the structural basis for such activation, an effect
that has been reported previously in the literature for
Scheme 1. Synthesis of 20, identified in the virtual screening as a
potential GS inhibitor, and two analogues (21– 22). (a) AcNHCH
(CO2Et)2, NaOEt, EtOH; (b) H2, Pd/C, 1 atm, HCl, EtOH, CHCl3; (c)
NaNO2, H2O, reflux; (d) i—NEt3, MsCl, THF; ii—NaI, acetone; (e)
P(OEt)3; (f) 9 M HCl.

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
Table 3. Synthetic library based on 23 identified in the virtual screening
HOOC
5507
R1
HN
HOOC
HN
OH
O
S
R2
O
23
OH
R2=
R1=
R1=
R1=
Compound
H
OH
OH
Compound
% Inhibitiona
AAb
Compound
% Inhibitiona
% Inhibitiona
O
24
29
AAb
34
AAb
OH
NIb,c
30
31
AAb,d
35
36
NIb,d
NIb
S
S
25
26
O
30 18b
13 15b
O
S
O
27
28
NIb
32
33
AAb
AAb
37
38
33 10b
NH2
O
42 5e
AAb
P
OEt
OEt
Inhibition of MtGS is reported as percent inhibition at a concentration of 1.0 mM, as compared to uninhibited enzyme. Substrate concentrations in
the assay were 1 mM ATP, 10 mM L-glutamate and 30 mM ammonium chloride. NI indicates no inhibition and AA indicates apparent activation.
a Values are an average from three independent experiments, reported together with the standard deviation.
b Dissolved in H2O.
c Concentration in assay 0.90 mM.
d Concentration in assay 0.50 mM.
e Dissolved in DMSO.
were particularly potent against MtGS, suggesting that
the amino acid binding site may not be the best focus
for future efforts. Recent structural information50 indi-
cates that the less-conserved nucleotide-binding site could
instead be explored as a means of attaining the desired
specificity for the mycobacterial as compared to the mam-
malian enzyme.
Scheme 2. Procedure for the synthesis of a library based on 23
originating from the virtual screening. (a) ArBr, amino acid, 10 mol%
CuI in water–acetonitrile, 90 ꢁC.
4. Experimental
4.1. General information
plant GSs.30 However, to the best of our knowledge, acti-
vation of a bacterial GS has not been described.
1H NMR and 13C NMR spectra were obtained on a
Varian Mercury 400 spectrometer (1H 400 MHz, 13C
100.6 MHz). Chemical shifts are reported as d values
(ppm) referenced to TMS via the solvent signal. GC–
MS analyses were performed with a CP-SIL 8 CB
Low Bleed (30 m · 0.25mm) or a CP-SIL 5 CB Low
Bleed (30 m · 0.25 mm) capillary column using a 40–
300 ꢁC temperature gradient and EI ionization. HPLC-
MS was performed on a Gilson HPLC system with a
Finnigan AQA quadropole mass spectrometer, using
an Onyx Monolithic C18 column (50 · 4.6 mm) at a flow
rate of 4 mL/min and a H2O/CH3CN/0.05% HCOOH
gradient; detection was by UV (DAD) and MS
(ESI+). Preparative RP-HPLC was performed by
UV-triggered fraction collection with a similar Gilson-
Finnigan AQA system and a Zorbax SB C8 column
(150 · 21.2 mm) at a flow rate of 10–12 mL/min and a
3. Conclusions
By using a literature survey, virtual screening and synthe-
sis of analogues, ten new compounds inhibiting MtGS
have been identified. Hits were identified by all three ap-
proaches. The best compound identified (4) had an IC50
of 610 15 lM, and has previously been reported as an
inhibitor of S. typhimurium GS,29 and to a lesser extent,
sheep brain and soybean GS.35 Three novel inhibitors
with aromatic scaffolds were found among the virtual
screening compounds. These compounds (14, 15 and 23)
showed 24–48% inhibition at 1.0 mM, respectively.
Among the synthesized N-arylated amino acids, one
inhibitor (28) was identified with 42% inhibition at
1.0 mM. However, we note that none of the inhibitors

5508
A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
H2O/CH3CN/0.05% HCOOH gradient. Chiral HPLC
analysis was performed using an AGP 150.4 column
(150 · 4.0 mm) using a phosphate buffer (pH 5.5) and
2–3% 2-propanol as solvent (flow rate 1 mL/min).
unadenylylated enzyme. The protein was purified and
stored as previously described.44
4.2.2. Assay development. Quantification of released
phosphate in the assay was performed using a commercial
inorganic phosphate detection reagent that produces a
green colour with an absorption maximum at 635 nm.
The reaction (100 lL) was stopped by addition of PiLock
Gold reagent (25 lL, PiLock reagent mixed 1:100 with
accelerator reagent). After 5 min incubation at room tem-
perature, stabilizer (10 lL) was added to the solution, and
an additional 30 min incubation at room temperature
was performed before measuring the absorbance at
635 nm using a microtitre plate reader. A linear response
was observed from 1.6 lM to 50 lM, in agreement with
the manufacturer’s specifications. The highest Pi concen-
tration tested, 100 lM, deviated from linearity, due to the
precipitation of the developed coloured compound.
Column chromatography was performed using silica
gel 60 (particle size 0.040–0.063 mm, E. Merck). For
reversed phase flash chromatography, silica gel RP-18
(0.040–0.063 mm, E. Merck) was used. Thin-layer chro-
matography was performed with aluminium sheets
coated with silica gel 60 F254 (0.2 mm E. Merck), using
UV-light or iodine vapour for visualization. Optical
rotations were obtained at room temperature on a
polarimeter; specific rotations ([a] D) are reported in
deg/dm and the concentration (c) is given in g/100 mL
in the specified solvent. THF was freshly distilled over
˚
Na/benzophenone. Acetone was dried over 3 A molecu-
lar sieves. All products were >95% pure according to
either GC–MS or LC-MS (TIC).
For enzyme assays, GS was diluted to 3.6 ng/lL
(approximate subunit concentration of 70 nM) into
HEPES–HCl (100 mM), pH 7.5, MgCl2 (50 mM) and
equilibrated at room temperature for at least 30 min-
utes. The assay was subsequently performed in
HEPES–HCl (50 mM), pH 7.5, MgCl2 (25 mM), ATP
(1 mM), NH4Cl (30 mM) and monosodium L-glutamate
(10 mM). The reaction was initiated by the addition of
glutamate (50 lL), resulting in a final volume of
100 lL and an enzyme subunit concentration of 7 nM.
The assay was performed in a 96-well microtitre plate
format and incubated at room temperature.
All other reactants and reagents were commercially
available and used without further purification unless
otherwise stated. L-Methionine-(S)-sulfoximine (1) and
(2S,5R)-2,6-diamino-5-hydroxyhexanoic acid (4) were
purchased from Fluka. DL-Phosphinothricin (2) was
purchased from Duchefa Biochemie, Haarlem, The
Netherlands. L-Methionine sulfone (7), L-methionine-
( SR)-sulfoxide (8), L-2-amino-4-sulfobutyric acid (13)
and (23) were purchased from Sigma Chemical Co.,
St. Louis, MO USA. L-Homoserine (10) was purchased
from Novabiochem, Merck KGaA, Darmstadt, Ger-
many. Compounds (6, 16–19) were purchased from
InterBio Screen.51 Compounds 6, 16–18 were purchased
as L-amino acids and 19 was supplied as a racemic
mixture. Compound 14 was purchased from Asinex.52
Compound 15 was purchased from Chemdiv.53 The
structure of the purchased virtual screening compounds
A linear increase in the amount of phosphate detected
was observed for 2 hours. Running the assay reaction
for a longer time would be expected to generate phos-
phate concentrations outside the linear range of the
detection system. Therefore, under standard conditions
during the inhibition study the assay was run for
60 min. To assess the quality of the results, Z0 was calcu-
lated as described previously by Zhang et al.56 A Z0-fac-
tor greater than 0.5 is regarded as an excellent outcome;
Z0-factors of 0.73 0.15 (H2O) and 0.69 0.19 (2.3%
(v/v) DMSO) were obtained here.
1
was confirmed by H NMR and HPLC-MS. L-2-Ami-
no-4-hydroxyaminobutyric acid (5),35 L-5-hydroxynorv-
aline (9)54 (2S,5S)-2,6-diamino-5-hydroxyhexanoic acid
(11),55 L-2-amino-4-(hydroxymethylamino)butyric acid
(12)34 and 2-amino-3-(2-phosphonomethylphenyl)prop-
anoic acid (20)48 are known compounds. Compound 24
is a known structure, where additional data are given.
Compounds 21, 22 and 25– 38 are new compounds. N-
arylated amino acids gradually decomposed at room
temperature, prohibiting accurate elemental analysis.
4.2.3. Initial inhibition studies. Compounds were dis-
solved in MilliQ water or in DMSO to prepare a
stock solution (50 mM) that was stored frozen at
ꢁ20 ꢁC. The compound (2 ll) was added to a micro-
titre plate and mixed with enzyme solution (50 ll)
containing all components except L-glutamate. The
enzyme–compound–substrate mixture was then pre-
incubated for 20 min at room temperature prior to
addition of L-glutamate to start the reaction (50 ll,
20 mM). The final compound concentration after
addition of glutamate was 1 mM. For reactions with
compounds dissolved in DMSO the final concentra-
tion of solvent was 2% (v/v).
Stocks of ATP (100 mM) were prepared, neutralized by
sodium hydroxide and stored at ꢁ20 ꢁC. Phosphate
detection reagents (PiColorlock Gold) were purchased
from Innova Bioscience (Cambridge, UK. url: http://
www.innovabiosciences.com). Sodium L-glutamate was
purchased from Sigma–Aldrich, St. Louis, MO USA.
A stock solution was prepared using MilliQ water (Mil-
lipore) and the pH was adjusted to 7.5; aliquots were
stored at ꢁ20 ꢁC.
4.2. Biological testing
Each compound was assayed in triplicate and the results
reported as means together with a standard deviation.
The activity is presented as the percentage of inhibition.
The Student’s 2-sided T-test was used to determine if the
observed activity was significantly different from the
4.2.1. GS samples. MtGS was heterologously expressed
in a GlnE-deficient E. coli strain (a generous gift of
AstraZeneca India, Limited), which yields a completely

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
5509
uninhibited reaction. The p-value (p < 0.1) was used as
the threshold for scoring a compound as active.
Stereoisomers, tautomers and two ring conformations
were automatically generated and the ligands were ion-
ized and minimized with the force field OPLS2003. The
UNITY search yielded 46,400 compounds, which was
expanded to 102,379 structures to be used in the
docking calculations. Babel71 was used for file conver-
sion between sdf and mol2 format. The protein44
(PDB code 2BVC) was prepared with the protein
preparation and refinement feature implemented in
Maestro. ADP and MSO-P were removed, as well as
all water molecules.
To correct for an increased signal due to contaminating
phosphate in the compound sample, and for possible
intrinsic absorbance of the compound itself, a control
plate was used.57 Each compound (2 lL) was treated as
in the inhibition experiment except that GS and ATP were
excluded from the reaction. The signal resulting from the
phosphate contamination was subtracted from that ob-
tained in the inhibitory assay (ODcorr=ODassay
ꢁ
ODCBG, where ODassay is defined as the optical density
reading of the inhibition assay, and ODCBG as the optical
density reading of the compound background).
FlexX72,73 was used as a docking tool with default set-
tings. The active site was defined by the residues
Glu133, Glu135, Tyr186, Glu219, Glu227, Asn271,
Gly272, His276, Arg329, Glu335, Arg347, Arg352,
Glu366, Arg368, and Tyr406 from one subunit, as well
as Asp54 and Ser57 from a neighbouring molecule,
and the three Mg2+ ions. Only the highest ranked pose
in the docking was retained and subjected to a rigid
pharmacophore search (Fig. 3) in UNITY. The pharma-
cophore model that was built based on MSO consisted
of two hydrogen bond acceptors and one hydrogen
4.2.4. IC50 determination. To determine the IC50 values,
initial stock solutions of compound were prepared in
MilliQ water and serially diluted to an appropriate con-
centration range. The final compound concentrations
ranged from 1.2 mM with a 2-fold serial dilution to a
concentration of 4.7 lM (MSO 1), 1 mM to 1.5 lM
(PPT 2, 3-fold serial dilution) and 10 mM to 39 lM (4,
2-fold serial dilution). The assay was performed as pre-
viously described for the inhibition assay. Only com-
pounds that led to more than 50% inhibition at 1 mM
were evaluated. Three or more independent experiments
were carried out for each compound. IC50 values were
separately determined from each dilution series by non-
linear regression, fitting a three-parameter equation (Eq.
1) to the data from the microtitre plate readings at
635 nm, that is
˚
bond donor, with a tolerance criterion of 2 A. A partial
match constraint to fit at least two out of three pharma-
cophore points was employed. The remaining 3511 com-
pounds that fulfilled the pharmacophore criterion were
visually inspected and 29 compounds were selected for
purchase, based on how well they fulfilled the following
criteria: (a) a realistic conformation in the active site, (b)
a tetrahedral atom in close proximity to the sulfur atom
of MSO, (c) a negatively charged functional group in
place of the phosphate from MSO-P and (d) an overall
similarity to an amino acid. Compounds with large
hydrophobic substituents were not selected.
Hi ꢁ Lo
IC50
Y ¼ Lo þ
ð1Þ
X
1 þ
where Hi is the estimated highest absorbance at zero
inhibitor concentration, Lo the estimated lowest absor-
bance at infinite inhibitor concentration, X the concen-
tration of inhibitor and Y the measured absorbance.
Regression analysis was performed using the SOLVER
function in the Microsoft Excel spreadsheet soft-
ware58–60 with the goal of maximizing the square of
the correlation coefficient (R2), and including the con-
straint that IC50 P 0. The reported IC50 values are an
average of those obtained from three (1 and 2) or five
(4) separate experiments together with the standard
deviation.
4.4. Chemistry
4.4.1. Diethyl (2-cyanobenzyl)acetamidomalonate (39).48
To a 21% solution of NaOEt in EtOH (10.5 mL,
28.1 mmol), diethyl acetamidomalonate (5.00 g, 23 mmol)
and 2-cyanobenzylbromide (5.01 g, 25.5 mmol) were
added successively. The reaction was stirred at room tem-
perature under nitrogen for 36 h. The precipitate was dis-
solved in MeOH, and dichloromethane (DCM) and
Celite were added before the solvent was removed under re-
duced pressure. Flash chromatography of the crude prod-
uct with DCM–acetone (19:1) gave 39 as a white solid
(4.7 g, 62% yield) with ethyl methyl (2-cyanobenzyl)ace-
tamidomalonate, approximately 12:1 as determined from
1H NMR, resulting from transesterification, as a sideprod-
uct. Experimental data were in accordance with literature
data.48
4.3. Virtual screening
A
database containing approximately 2.1 million
compounds was assembled.51–53,61–68 A 2D UNITY sub-
structure search69 was performed to identify compounds
containing carboxylic acid bioisosters43 (substrate ana-
logues), primary amides (product analogues) or func-
tional groups that have previously been used to
substitute for the sulfoximine group of the MtGS
inhibitor MSO29 (Fig. 3). The search was limited to
4.4.2. Diethyl [2-(aminomethyl)benzyl]acetamidomalonate
(40).48 To a suspension of 10% Pd on charcoal (1.22 g)
in EtOH (1.2 mL) was added a solution of 39 (3.7 g,
11 mmol) in CHCl3 (45 mL), EtOH (23 mL) and con-
centrated HCl (3.5 mL). The reaction was stirred under
H2 at 1 atm, 40 ꢁC for 24 h. After filtration and evap-
oration, the residue was taken up in water (20 mL)
and extracted with DCM (2 · 20 mL). From the organ-
ic phase unreacted 39 was recovered after evaporation.
compounds with
a
molecular weight less than
300 g/mol, due to the small size of the amino acid bind-
ing pocket. The ligprep module implemented in
Maestro70 was used for ligand pre-treatment steps. Salts
were removed and hydrogens were added to all ligands.

5510
A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
The water phase was freeze dried to obtain 40 (0.48 g,
12% yield) as a light yellow powder with ethyl methyl
[2-(aminomethyl)benzyl]acetamidomalonate as a side-
product. Experimental data were in accordance with
literature data.48
HPLC (45 min 0–50% CH3CN) and 20 was obtained as
white powder (11 mg, 10% yield). Experimental data were
in accordance with literature reports.48
4.4.7. 2-Amino-3-(2-aminomethylphenyl)propanoic acid
(21). Compound 40 (100 mg, 260 lM) was refluxed over
night in 9 M HCl (5 mL) and concentrated to dryness.
The crude product was purified using preparative HPLC
(45 min 0–30% CH3CN) and 21 was obtained as a green
solid (55 mg, 86% yield).
4.4.3. Diethyl [2-(hydroxymethyl)benzyl]acetamidomalo-
nate (41) .48 Compound 40 (1.5 g, 4.02 mmol) was dis-
solved in water (25 mL) and the pH was adjusted to 6
with 1 M NaOH. NaNO2 (0.405 g, 5.87 mmol) was added
and the reaction mixture was refluxed for 5 h, cooled and
extracted with EtOAc(4 · 25 mL). The combined organ-
ic phases were washed with 1 M HCl (10 mL), water
(10 mL), 5% NaHCO3 (10 mL), water (10 mL) and brine
(10 mL) and dried over Na2SO4. Flash chromatography
with EtOAc–isohexane (6:4) gave 41 as a yellow oil
(0.47 g, 34% yield) with ethyl methyl [2-(hydroxy-
1H NMR (D2O, 400 MHz): d 7.48–7.34, 4.25 (s, 2H),
4.13 (dd, J = 8.0, 6.8 Hz, 1H), 3.40 (dd, J = 14.9,
8.0 Hz, 1H), 3.22 (dd, J = 14.9, 6.8 Hz, 1H) 13C NMR
(D2O–CD3OD, 2:1, 100.5 MHz): d 174.3, 136.0, 132.6,
131.9, 131.4, 130.9, 129.4, 56.8, 41.0, 34.3. Anal. calcd
for C10H14N2O2 + HCl + 0.5 H2O: C, 50.11; H, 6.73;
N, 11.69; Cl, 14.79. Found: C, 49.79; H, 6.57; N,
11.52; Cl, 14.50.
methyl)benzyl]acetamidomalonate as
a sideproduct.
Experimental data were in accordance with literature
data.48
4.4.8. Methyl 2-amino-3-(2-phosphonomethylphenyl)pro-
panoate (22). Compound 22 was obtained in the depro-
tection of 20 and was easily separated using preparative
HPLC (45 min 0–50% CH3CN), which produced 22 as a
white powder (53 mg, 46% yield).
4.4.4. Diethyl [2-(iodomethyl)benzyl]acetamidomalonate
(42). To a solution of 41 (0.50 g, 1.48 mmol) in dry THF
(8 mL) at 0 ꢁC was added NEt3 (0.35 mL, 2.51 mmol)
and MsCl (0.2 mL, 2.58 mmol).74 The resulting mixture
was stirred for 1 h and quenched with 10% (w/w) citric
acid (10 mL), extracted with EtOAc (3 · 25 mL) and
washed with brine (10 mL). The organic phase was dried
over MgSO4, filtered and evaporated. Crude diethyl [2-
(methanesulfonyloxymethyl)benzyl]acetamidomalonate
was directly dissolved in dry acetone (10 mL). NaI
(0.907 g, 6.05 mmol) was added and the reaction mix-
ture was stirred at room temperature for 2 h.74 The reac-
tion solvent was removed and the residue taken up in
EtOAc (40 mL). The organic layer was washed sequen-
tially with 10% (w/w) citric acid (30 mL), 10% (w/w)
Na2S2O3 (30 mL) and dried over MgSO4. Evaporation
of the solvent gave 42 as a yellow oil (0.50 g, 75% yield)
with ethyl methyl [2-(iodomethyl)benzyl]acetamidomal-
onate as a sideproduct. The product was used directly
in the next step without further purification.
1H NMR (D2O, 400 MHz): d 7.39–7.22, 4.49 (m, 1H),
3.86 (s, 3H), 3.50 (dd, J = 14.8, 9.0 Hz, 1H), 3.30 (dd,
J = 14.8, 5.8 Hz, 1H), 3.10 (m, 2H) 13C NMR (D2O–
CD3OD, 2:1, 100.5 MHz):
d
171.5, 135.7 (d,
J = 8.7 Hz), 133.4 (d, J = 5.9 Hz), 132.9 (d, J = 5.1 Hz),
130.6 (d, J = 3.1 Hz), 128.9 (d, J = 3.2 Hz), 128.0 (d,
J = 3.6 Hz), 54.5, 54.4, 34.2 (d, J = 128 Hz), 33.2. Anal.
calcd for C11H16NO5P+0.5 H2O: C, 46.81; H, 6.07; N,
4.96. Found: C, 46.97; H, 5.90; N, 4.84.
4.4.9. (SR)-1-Bromo-3-methanesulfinylbenzene (44) .75
Phenol (5.2 g, 55.3 mmol) was melted at 45 ꢁC and
mixed
with
1-bromo-3-methylsulfanyl-benzene
(0.4 mL, 2.98mmol). Afterwards 30% (w/w) aq H2O2
was added (0.56 mL) and the reaction mixture was stir-
red for 30 s. Excess H2O2 was quenched with saturated
Na2SO3 solution (20 mL) and the pH was adjusted to
10 with 1 M NaOH. The water phase was extracted
with EtOAc (3 · 30 mL) and the combined organic
phases were washed with water (30 mL), brine
(30 mL) and dried over Na2SO4. The solvent was re-
moved and 44 was obtained after flash chromatogra-
phy with petrol ether–EtOAc (6:1 ! 1:2) as white
crystals (627 mg, 96% yield).
1H NMR (CDCl3, 400 MHz): d 7.36 (m, 1H), 7.16 (m,
2H), 6.93 (m, 1H), 4.43 (s, 2H), 4.30 (dq, J = 10.7, 7.2
Hz, 2H), 4.23 (dq, J = 10.7, 7.2 Hz, 2H), 3.75 (s, 2H),
2.00 (s, 3H), 1.28 (t, J = 7.2 Hz, 6H) 13C NMR (CDCl3,
100.5 MHz): d 169.5, 167.6, 138.9, 133.2, 130.9, 130.8,
128.0, 127.9, 66.6, 62.8, 34.4, 29.7, 23.1, 13.9.
4.4.5. Diethyl [2-((diethoxyphospinyl)methyl)benzyl]ace-
tamidomalonate (43).48 Compound 42 (0.50 g, 1.12
mmol) was dissolved in 5 mL P(OEt)3, refluxed overnight
and concentrated in vacuo. Flash chromatography with
isohexane–EtOAc–MeOH (6:4:0.5) gave 43 as a yellow
oil (0.25 g, 49% yield) with ethyl methyl [2-((diethoxyp-
hospinyl)methyl)benzyl]acetamidomalonate as a side-
product. Experimental data were in accordance with
literature data.48
[a] D: 0.00 Rf (petrol ether–EtOAc, 2:1, v/v) 0.52. Exper-
imental data were in accordance with literature data.76
4.4.10. (3-Bromophenyl)phosphonic acid diethyl ester (45).77
To a mixture of palladium acetate (354 mg, 1.56 mmol)
and 3-bromoiodobenzene (2.54 mL, 19.9 mmol) was
added P(OEt)3 (3.6 mL, 21 mmol). The reaction mixture
was stirred at 90 ꢁC for 20 h. After cooling, diethylether
(20 mL) was added and the mixture was filtered. The sol-
vent was removed under reduced pressure and flash chro-
matography with DCM–CH3OH (200:1 ! 100:1) gave
45 as a colourless oil (4.4 mg, 75%).
4.4.6. 2-Amino-3-(2-phosphonomethylphenyl)propanoic acid
(20).48 Compound 43 (0.186 g, 0.41 mmol) was refluxed
overnight in 9 M HCl (6.6 mL) and concentrated to dry-
ness. The crude product was purified using preparative

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
5511
Rf (DCM–CH3OH, 19:1, v/v) 0.51. Experimental data
were in accordance with literature data.78
(s, 2H), 1.92 (s, 2H). HRMS (M+1): Calcd: 231.0440
Found: 231.0435.
4.4.11. General procedure for the N-arylation of amino
acids. Synthesis of 24–38. Aryl bromide (1 mmol), CuI
(19.5 mg, 0.1 mmol) and K2CO3 (166 mg, 1.2 mmol)
were dissolved in a CH3CN–H2O mixture ([1:4,v/v]
2.5 mL). The amino acid (2 mmol) was added and the
reaction mixture was stirred for 24–50 h at 90 ꢁC. The
reaction was cooled (0 ꢁC), diluted with DCM (20 mL)
and H2O (20 mL) and the pH was adjusted to 2–3 with
concd HCl. The water phase was extracted with DCM
(2· 30 mL) and the combined organic phases were
washed with brine (20 mL). Either the product was
found in the water phase, which was concentrated and
the crude product was isolated after freeze drying, or
the product was found the organic phase, which was
dried over MgSO4 and evaporation of the solvent
yielded the crude product. The crude product was puri-
fied by with preparative RP-HPLC (30 min 10–60% or
80% CH3CN).
4.4.16. [3-(Diethoxyphosphoryl)phenylamino]acetic acid
(28). Following the general procedure, 45 (0.1 mmol)
was N-arylated with glycine to give 5.8 mg (20.2%) of
28 after preparative HPLC.
1H NMR (CD3CN, 400 MHz): d 7.07 (m, 1H), 6.97 (dt,
J = 1.6, 14.9 Hz, 1H), 6.85 (dd, J = 7.4, 12.9 Hz, 1H),
6.76 (d, J = 8.2 Hz, 1H), 4.0–3.84 (m, 6H), 3.22 (s,
1H), 1.15 (t, J = 7.0 Hz, 6H). HRMS (M+1): Calcd:
288.1001 Found: 288.1006.
4.4.17. 3-((S)-1-Carboxy-2-hydroxyethylamino)benzoic
acid (29). Following the general procedure, 3-bromoben-
zoic acid (0.1 mmol) was N-arylated with L-serine and
29 (5.1 mg, 22.7%) was obtained after preparative
HPLC.
1H NMR (D2O, 400 MHz): d 7.60–6.90 (m, 4H), 4.58–
3.79 (m, 4H). HRMS (M+1): Calcd: 226.0715 Found:
20
D
4.4.12. 3-(Carboxymethylamino)benzoic acid (24). Fol-
lowing the general procedure, 3-bromobenzoic acid
(0.1 mmol) was N-arylated with glycine. Compound 24
(6.2 mg, 31.8%) was obtained after preparative HPLC.
226.0721. ½aꢂ 45.6 (c 1.2, H2O).
4.4.18.
(S)-3-Hydroxy-2-(3-methylsulfanylphenylami-
no)propionic acid (30). Following the general procedure,
3-bromothioanisole (0.05 mmol) was N-arylated with
L-serine to obtain 2.0 mg (17.6%) of 30 after preparative
HPLC.
1H NMR (D2O, 400 MHz): d 7.60–7.40 (m, 3H), 7.15
(m, 1H), 4.06 (s, 2H). HRMS (M+1): Calcd: 196.0610
Found: 196.0614. Experimental data were in accordance
with literature data.79
1H NMR (CDCl3, 400 MHz): d 6.62 (t, J = 7.4 Hz, 3H),
6.54 (d, J = 8.1 Hz, 1H), 4.88 (s, 1H), 3.88–3.52 (m, 3H),
3.15 (s, 1H), 2.18 (s, 3H). HRMS (M+1): Calcd:
4.4.13. (3-Methylsulfanylphenylamino)acetic acid (25).
Following the general procedure, 3-bromothioani-
sole (0.05 mmol) was N-arylated with glycine to
obtain 2.8 mg (28.4%) of 25 after preparative
HPLC.
20
228.0694 Found: 228.0690.½aꢂ ꢁ67 (c 0.1, CHCl3).
D
4.4.19.
(S)-3-Hydroxy-2-(3-methanesulfinylphenylami-
no)propionic acid (31). Following the general procedure,
44 (0.1 mmol) was N-arylated with L-serine. Compound
31 (4.8 mg, 19.8%) was obtained after preparative
HPLC.
1H NMR ((CD3)2CO, 400 MHz): d 7.05 (t, J = 7.8 Hz,
1H), 6.59 (s, 1H), 6.56 (d, J = 7.8 Hz, 1H), 6.46 (dd,
J = 1.4, 8.2 Hz, 1H), 3.92 (s, 2H), 3.92 (s, 1H), 2.42 (s,
3H). 13C NMR ((CD3)2CO, 100.5 MHz): d 206.4,
172.4, 140.1, 130.2, 116.0, 111.2, 110.5, 45.6, 15.4.
HRMS (M+1): Calcd: 198.0589 Found: 198.0595.
1H NMR (CDCl3, 400 MHz): d 6.92 (t, J = 7.1 Hz, 1H),
6.55 (s, 1H), 6.53 (t, J = 6.0 Hz, 1H), 6.39 (d, J = 7.4 Hz,
1H), 4.73 (s, 1H), 3.79 (s, 1H), 3.60 (s, 2H), 3.20 (s, 1H),
2.36 (s, 1H). HRMS (M+1): Calcd: 244.0644 Found:
244.0652.
4.4.14. (3-Methanesulfinylphenylamino)acetic acid (26).
Following the general procedure, 44 (0.1 mmol) was
N-arylated with glycine, and 26 (5.0 mg, 23.5%) was
obtained after preparative HPLC.
4.4.20. (S)-3-Hydroxy-2-(3-sulfamoylphenylamino)propi-
onic acid (32). Following the general procedure, 3-
bromobenzenesulfonamide (0.1 mmol) was N-arylated
with L-serine to obtain 14 mg (53.8%) of 32 after pre-
parative HPLC.
1H NMR (CDCl3, 400 MHz): d 7.09 (t, J = 7.8 Hz, 1H),
6.97 (t, J = 7.8 Hz, 1H), 6.68 (t, J = 7.4 Hz, 1H), 6.51
(dd, J= 2.0, 8.1 Hz, 1H), 3.74 (s, 2H) 2.97 (s, 1H), 2.51
(s, 3H). HRMS (M+1): Calcd: 214.0538 Found:
214.0533.
1H NMR ((CD3)2CO, 400 MHz): d 6.82 (s, 1H), 6.72–
6.40 (m, 2H), 6.30–6.20 (m, 1H), 3.48 (s, 2H), 2.94 (s,
1H), 1.58 (s, 2H). Calcd: 261.0545 Found: 261.0550.
20
D
4.4.15. (3-Sulfamoylphenylamino)acetic acid (27). Fol-
lowing the general procedure, 3-bromobenzenesulfona-
mide (0.1 mmol) was N-arylated with glycine to
produce 12 mg (52.2%) of 27 after preparative HPLC.
½aꢂ 14 (c 0.5, CHCl3).
4.4.21.
(S)-2-[3-(Diethoxyphosphoryl)phenylamino]-3-
hydroxypropionic acid (33). Following the general pro-
cedure, 45 (0.1 mmol) was N-arylated with L-serine to
obtain 20.4 mg (64.4%) of 33 after preparative
HPLC.
1H NMR (CDCl3, 400 MHz): d 7.14 (t, J = 7.0 Hz, 1H),
6.99 (s, 1H), 6.70 (s, 1H), 6.42 (s, 1H), 5.80 (s, 1H), 3.85

5512
A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
1H NMR (CD3CN, 400 MHz): d 7.26 (m, 1H), 7.12 (d,
J = 14.6 Hz, 1H), 7.03 (dd, J = 6.4, 12.9 Hz, 1H), 6.92
(d, J = 7.8 Hz, 1H), 5.34 (s, 1H), 4.23 (t, J = 4.4 Hz,
1H), 4.08–3.96 (m, 6H), 3.55 (s, 1H), 1.26 (t,
Acknowledgments
We thank the Swedish Foundation for Strategic
Research (SSF), the Swedish Research Council (VR),
the Knut and Alice Wallenberg Foundation and the
EU Sixth Framework Program NM4TB CT:01892 for
financial support. We express our sincere gratitude to
AstraZeneca India Pvt Limited for providing the GlnE-
deficient E. coli strain. We also thank Dr. Aleh Yahorau
for his kind help with the high-resolution mass analysis.
J = 7.0 Hz, 6H). HRMS (M+1): Calcd: 318.1107 Found:
20
D
318.1101. ½aꢂ 106 (c 0.4, CHCl3).
4.4.22. 3-((R)-1-Carboxy-2-hydroxyethylamino)benzoic
acid (34). Following the general procedure, 3-bromo-
benzoic acid (0.1 mmol) was N-arylated with D-serine
to obtain 5.5 mg (24.4%) of 34 after preparative
HPLC.
1H NMR (CDCl3, 400 MHz): d 6.92–6.75 (m, 3H), 6.38
(s, 1H), 4.80 (s, 2H), 3.64–3.44 (m, 2H), 2.16 (m, 1H).
References and notes
20
D
1. O’Brien, R. J.; Nunn, P. P. Am. J. Respir. Crit. Care Med.
2001, 163, 1055.
2. Duncan, K. Tuberculosis 2003, 83, 201.
3. Liaw, S. H.; Eisenberg, D. Biochemistry 1994, 33, 675.
4. Harth, G.; Clemens, D. L.; Horwitz, M. A. Proc. Natl.
Acad. Sci. U.S.A. 1994, 91, 9342.
5. Sassetti, C. M.; Boyd, D. H.; Rubin, E. J. Mol. Microbiol.
2003, 48, 77.
6. Harth, G.; Zamecnik, P. C.; Tang, J. Y.; Tabatadze, D.;
Horwitz, M. A. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
418.
7. Tullius, M. V.; Harth, G.; Horwitz, M. A. Infect. Immun.
2003, 71, 3927.
8. Harth, G.; Horwitz, M. A. J. Exp. Med. 1999, 189, 1425.
9. Harth, G.; Horwitz, M. A. Infect. Immun. 2003, 71, 456.
10. Borek, E.; Miller, H. K.; Sheiness, P.; Waelsch, H. J. Biol.
Chem. 1946, 163, 347.
11. Ronzio, R. A.; Rowe, W. B.; Meister, A. Biochemistry
1969, 8, 1066.
12. Rowe, W. B.; Ronzio, R. A.; Meister, A. Biochemistry
1969, 8, 2674.
HRMS (M+1): Calcd: 226.0715 Found: 226.0708. ½aꢂ
ꢁ50.6 (c 1.1, H2O).
4.4.23.
(R)-3-Hydroxy-2-(3-methylsulfanylphenylami-
no)propionic acid (35). Following the general procedure,
3-bromothioanisole (0.05 mmol) was N-arylated with D-
serine to obtain 1.2 mg (10.6%) of 35 after preparative
HPLC.
1H NMR (CDCl3, 400 MHz): d 6.62 (t, J = 7.4 Hz, 3H),
6.54 (d, J = 8.1 Hz, 1H), 4.88 (s, 1H), 3.88–3.52 (m, 3H),
3.15 (s, 1H), 2.18 (s, 3H). M = 227.28 g/mol MS m/z
20
(70 eV) 228 (M+H+). ½aꢂ ꢁ60 (c 0.1, CHCl3).
D
4.4.24. (R)-3-Hydroxy-2-(3-methanesulfinylphenylami-
no)propionic acid (36). Following the general procedure,
44 (0.1 mmol) was N-arylated with D-serine. Compound
36 (5.3 mg, 21.8%) was obtained after preparative
HPLC.
13. Wedler, F. C.; Horn, B. R. J. Biol. Chem. 1976, 251, 7530.
14. Wedler, F. C.; Horn, B. R.; Roby, W. G. Arch. Biochem.
Biophys. 1980, 202, 482.
15. Wedler, F. C.; Sugiyama, Y.; Fisher, K. E. Biochemistry
1982, 21, 2168.
1H NMR (CDCl3, 400 MHz): d 6.93 (t, J= 7.2 Hz, 1H),
6.57 (s, 1H), 6.52 (t, J = 6.1 Hz, 1H), 6.39 (d, J = 7.4 Hz,
1H), 5.14 (s, 1H), 3.79 (s, 1H), 3.60 (s, 2H), 3.12 (s, 1H),
2.36 (s, 1H). HRMS (M+1): Calcd: 244.0644 Found:
244.0649.
16. Farrington, G. K.; Kumar, A.; Wedler, F. C. J. Med.
Chem. 1987, 30, 2062.
17. Meek, T. D.; Villafranca, J. J. Biochemistry 1980, 19, 5513.
18. Harth, G.; Griffith, O. W.; Horwitz, M. A. WO 2004/
045539, 2004.
19. Berlicki, L.; Obojska, A.; Forlani, G.; Kafarski, P. J. Med.
Chem. 2005, 48, 6340.
4.4.25. (R)-3-Hydroxy-2-(3-sulfamoylphenylamino)propi-
onic acid (37). Following the general procedure,
3-bromobenzenesulfonamide (0.1 mmol) was N-arylated
with D-serine to obtain 9.5 mg (36.5%) of 37 after pre-
parative HPLC.
20. Forlani, G.; Obojska, A.; Berlicki, L.; Kafarski, P. J.
Agric. Food Chem. 2006, 54, 796.
21. Logusch, E. W. Tetrahedron Lett. 1988, 29, 6055.
22. Logusch, E. W.; Walker, D. M.; Mcdonald, J. F.; Leo, G.
C.; Franz, J. E. J. Org. Chem. 1988, 53, 4069.
23. Logusch, E. W.; Walker, D. M.; Mcdonald, J. F.; Franz,
J. E. Biochemistry 1989, 28, 3043.
24. Logusch, E. W.; Walker, D. M.; Mcdonald, J. F.; Franz,
J. E.; Villafranca, J. J.; Diianni, C. L.; Colanduoni, J. A.;
Li, B.; Schineller, J. B. Biochemistry 1990, 29, 366.
25. Logusch, E. W.; Walker, D. M.; Mcdonald, J. F.; Franz,
J. E. Plant Physiol. 1991, 95, 1057.
26. Walker, D. M.; Mcdonald, J. F.; Franz, J. E.; Logusch, E.
W. J. Chem. Soc., Perkin Trans. 1 1990, 659.
27. Johnson, C. R.; Boettcher, B. R.; Cherpeck, R. E.;
Dolson, M. G. Bioorg. Chem. 1990, 18, 154.
28. Lejczak, B.; Starzemska, H.; Mastalerz, P. Experientia
1981, 37, 461.
1H NMR ((CD3)2CO–CDCl3, 1:1, 400 MHz): d 6.87 (s,
1H), 6.73 (s, 1H), 6.42 (s, 1H), 6.18 (s, 1H), 4.0–3.40 (m,
3H), 1.69 (s, 2H). HRMS (M+1): Calcd: 261.0545
20
D
Found: 261.0541. ½aꢂ ꢁ5 (c 0.4, CHCl3).
4.4.26.
(R)-2-[3-(Diethoxyphosphoryl)phenylamino]-3-
hydroxypropionic acid (38). Following the general proce-
dure, 45 (0.1 mmol) was N-arylated with D-serine to ob-
tain 10.2 mg (32.2%) of 38 after preparative HPLC.
1H NMR (CD3CN, 400 MHz): d 7.26 (m, 1H), 7.12 (d,
J = 13.8 Hz, 1H), 7.02 (dd, J = 6.3, 12.8 Hz, 1H), 6.95
(d, J = 7.8 Hz, 1H), 4.27 (t, J = 4.4 Hz, 1H), 4.10–3.96
(m, 6H), 3.50 (s, 1H), 1.25 (t, J = 7.0 Hz, 6H). HRMS
20
D
(M+1): Calcd: 318.1107 Found: 318.1111. ½aꢂ ꢁ95 (c
29. Eisenberg, D.; Gill, H. S.; Pfluegl, G. M. U.; Rotstein, S.
H. Biochem. Biophys. Acta 2000, 1477, 122.
1, CHCl3).

A. Nordqvist et al. / Bioorg. Med. Chem. 16 (2008) 5501–5513
5513
30. Evstigneeva, Z. G.; Solov’eva, N. A.; Sidel’nikova, L. I.
Appl. Biochem. Microbiol. 2003, 39, 539.
53. ChemDiv Inc., 11558 Sorrento Valley Road, San Diego,
CA 92121, USA.
31. Acaster, M. A.; Weitzman, P. D. J. FEBS 1985, 189, 241.
32. Obojska, A.; Berlicki, L.; Kafarski, P.; Lejczak, B.;
Chicca, M.; Forlani, G. J. Agric. Food Chem. 2004, 52,
3337.
54. Hannachi, J. C.; Vidal, J.; Mulatier, J. C.; Collet, A. J.
Org. Chem. 2004, 69, 2367.
55. Allevi, P.; Anastasia, M. Tetrahedron Asymm. 2004, 15,
2091.
33. Leason, M.; Cunliffe, D.; Parkin, D.; Lea, P. J.; Miflin, B.
J. Phytochemistry 1982, 21, 855.
56. Zhang, J. H.; Chung, T. D.; Oldenburg, K. R. J. Biomol.
Screen. 1999, 4, 67.
34. Logusch, E. W.; Dukesherer, D. R.; Getman, K. M.;
Franz, J. E. Tetrahedron Lett. 1991, 32, 2711.
35. Fushiya, S.; Maeda, K.; Funayama, T.; Nozoe, S. J. Med.
Chem. 1988, 31, 480.
57. Singh, U.; Sarkar, D. J. Biomol. Screen. 2006, 11, 1035.
58. Brown, A. M. Comp. Meth. Prog. Biol. 2001, 65, 191.
59. Bowen, W. P.; Jerman, J. C. Trends Pharmacol. Sci. 1995,
16, 413.
36. Langston-Unkefer, P. L.; Macy, P. A.; Durbin, R. D.
Plant Physiol. 1984, 76, 71.
60. Parsons, J. D. Anal. Biochem. 2007, 360, 309.
61. ChemBridge Corporation, 16981 Via Tazon, Suite G, San
Diego, CA 92127, USA.
62. Enamine Ltd, 23 Alexandra Matrosova Street, 01103
Kiev, Ukraine.
63. Key Organics Ltd, Highfield Industrial Estate, Camelford,
Cornwall PL32 9QZ, UK.
64. Life Chemicals Inc., 2477 Glenwood School Drive Suite
203, Burlington, ON, Canada L7R 3R9.
65. Maybridge, Trevillet, Tintagel, Cornwall PL34 OHW,
UK.
37. Keseru, G. M.; Makara, G. M. Drug Discov. Today 2006,
¨
11, 741.
38. Bleicher, K. H.; Bohm, H. J.; Muller, K.; Alanine, A. I.
Nat. Rev. Drug Discov. 2003, 2, 369.
39. Liaw, S. H.; Kuo, I. C.; Eisenberg, D. Protein Sci. 1995, 4,
2358.
40. Manning, J. M.; Moore, S.; Rowe, W. B.; Meister, A.
Biochemistry 1969, 8, 2681.
41. Shrake, A.; Ginsburg, A.; Wedler, F. C.; Sugiyama, Y. J.
Biol. Chem. 1982, 257, 8238.
42. Lagerlund, O.; Odell, L. R.; Mowbray, S. L.; Nilsson, M.;
66. Sigma–Aldrich, 3050 Spruce Street, St. Louis, MO 63103,
USA.
´
Krajewski, W. W.; Nordqvist, A.; Karlen, A.; Larhed, M.
67. Specs, Kluyverweg 6, 2629 HT, Delft, The Netherlands.
68. Vitas-M Laboratory Ltd.
Comb. Chem. High Throughput Screen. 2007, 10, 783.
43. Stefanic, P.; Dolenc, M. S. Curr. Med. Chem. 2004, 11,
945.
44. Krajewski, W. W.; Jones, T. A.; Mowbray, S. L. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 10499.
45. Gill, H. S.; Eisenberg, D. Biochemistry 2001, 40, 1903.
46. Levintow, L.; Meister, A. J. Am. Chem. Soc. 1953, 75,
3039.
47. Wellner, V. P.; Zoukis, M.; Meister, A. Biochemistry 1966,
5, 3509.
48. Dorville, A.; McCort-Tranchepain, I.; Vichard, D.; Sath-
er, W.; Maroun, R.; Ascher, P.; Roques, B. P. J. Med.
Chem. 1992, 35, 2551.
49. Zhang, H.; Cai, Q.; Ma, D. W. J. Org. Chem. 2005, 70,
5164.
50. Krajewski, W. W.; Collins, R.; Holmberg-Schiavone, L.;
Jones, T. A.; Karlberg, T.; Mowbray, S. L. J. Mol. Biol.
2008, 375, 217.
51. InterBioScreen Ltd, PO Box 218, 119019 Moscow, Russia.
52. Asinex Ltd, 5 Gabrichevskogo, St. Bldg 8, Moscow
125367, Russia.
69. Unity Chemical Information Software, 4.4.1, Tripos Inc.,
1699 South Hanley Road, St. Louis, MO 63144, USA.
70. Maestro, 6.5, Schro¨dinger LLC., 120 West 45th Street,
32nd floor, New York, NY 10036-4041, USA.
71. Babel, 1.6, Walters, P.; Stahl, M., http://www.ccl.net/cca/
software/UNIX/babel/index.html.
72. Rarey, M.; Kramer, B.; Lengauer, T.; Klebe, G. J. Mol.
Biol. 1996, 261, 470.
73. FlexX, 1.13.5, Tripos Inc., 1699 South Hanley Road, St.
Louis, MO 63144, USA.
74. Dugave, C. J. Org. Chem. 1995, 60, 601.
75. Xu, W. L.; Li, Y. Z.; Zhang, Q. S.; Zhu, H. S. Synthesis
2004, 227.
76. Capozzi, M. A. M.; Cardellicchio, C.; Naso, F.; Tortorel-
la, P. J. Org. Chem. 2000, 65, 2843.
77. Villemin, D.; Elbilali, A.; Simeon, F.; Jaffres, P. A.;
Maheut, G.; Mosaddak, M.; Hakiki, A. J. Chem. Res.
2003, 436.
78. Bunnett, J. F.; Shafer, S. J. J. Org. Chem. 1978, 43, 1873.
79. Lu, Z.; Twieg, R. J. Tetrahedron Lett. 2005, 46, 2997.